2024 Volume 49 Issue 3 Pages 95-103
This study was conducted as part of an investigation into the cause of vesnarinone-associated agranulocytosis. When HL-60 cells were exposed to vesnarinone for 48 hr, little cytotoxicity was observed, although reduced glutathione (GSH) content decreased in a concentration-dependent manner. Significant cytotoxicity and reactive oxygen species (ROS) production were observed when intracellular GSH content was reduced by treatment with L-buthionine-(S, R)-sulphoximine. The involvement of myeloperoxidase (MPO) metabolism was suggested, as when HL-60 cells were exposed to a reaction mixture of vesnarinone–MPO/H2O2/Cl−, cytotoxicity was also observed. In contrast, the presence of GSH (1 mM) protected against these cytotoxic effects. Liquid chromatography–mass spectrometry analysis of the MPO/H2O2/Cl− reaction mixture revealed that vesnarinone was converted into two metabolites, (4-(3,4-dimethoxybenzoyl)piperazine [Metabolite 1: M1] and 1-chloro-4-(3,4-dimethoxybenzoyl)piperazine [Metabolite 2: M2]). M2 was identified as the N-chloramine form, a reactive metabolite of M1. Interestingly, M2 was converted to M1, which was accompanied by the conversion of GSH to oxidized GSH (GSSG). Furthermore, when HL-60 cells were exposed to synthetic M1 and M2 for 24 hr, M2 caused dose-dependent cytotoxicity, whereas M1 did not. Cells were protected from M2-derived cytotoxicity by the presence of GSH. In conclusion, we present the first demonstration of the cytotoxic effects and ROS production resulting from the MPO/H2O2/Cl− metabolic reaction of vesnarinone and newly identified the causative metabolite, M2, as the N-chloramine metabolite of M1, which induces cytotoxicity in HL-60 cells. Moreover, a protective role of GSH against the cytotoxicity was revealed. These findings suggest a possible nonimmunological cause of vesnarinone agranulocytosis.
Agranulocytosis is a life-threatening disease. It has been estimated that over 60% of all cases of agranulocytosis are caused by drugs (Kaufman et al., 1996). Although the incidence of drug-induced agranulocytosis is only 3.4 cases per million patients treated per year, this idiosyncratic drug-induced toxicity (IDT) is important because the incidence can reach as high as 1% in patients treated with certain drugs, such as clozapine (Alvir et al., 1993; Guest et al., 1998). Drug-induced agranulocytosis is characterized by a decrease in the peripheral neutrophil count to below 0.5 × 109 cells/L due to immunological or cytotoxic mechanisms (Pisciotta, 1990). Drugs associated with a significant incidence of idiosyncratic agranulocytosis are mainly bioactivated to reactive metabolites by myeloperoxidase (MPO), which is a major peroxidase in human neutrophils (Uetrecht, 1992). In general, MPO, along with hydrogen peroxide (H2O2) and chloride (Cl−), produces hypochlorous acid (HClO); this represents a potent neutrophil antimicrobial system. MPO is present in circulating neutrophil granulocytes (Maseneni et al., 2012) and in HL-60 cells, a human promyeloid cell line (Yamada et al., 1981). Therefore, HL-60 cells are frequently used in the investigation of agranulocytosis and neutropenia (Rudin et al., 2019; Torii-Goto et al., 2022).
Vesnarinone, a quinolinone derivative, was introduced in the late 1980s as an excellent positive inotropic agent for the treatment of symptomatic left ventricular dysfunction. However, in clinical trials, it was found that this drug induced agranulocytosis, with incidences of 0.2% at 30 mg/day and 1.2% at 60 mg/day (Cohn et al., 1998). Toxicity occurred within the first 30–90 days of treatment, gradually developing over several days, and spontaneously resolving within 10–14 days after discontinuation of the drug (Feldman et al., 1993). Uetrecht et al. (1994) studied the metabolism of vesnarinone by activated neutrophils, as well as the combination of MPO/H2O2/Cl− or HClO, and found evidence of a pathway that involved a reactive iminium ion. The authors demonstrated that hydrolysis of the iminium ion led to the formation of a reactive quinone imine. However, the significance of how these metabolites relate to agranulocytosis is not yet perfectly clear. Moreover, it has been reported that the inhibition of the differentiation of hematopoietic stem cells into mature granulocytes by vesnarinone, via stromal cell function, may cause the hematopoietic disorder of agranulocytosis (Aizawa et al., 1997). Bertolet (2004) compiled information on the cause of agranulocytosis using samples from patients who developed agranulocytosis after vesnarinone administration and unaffected patients. From this study, some information was obtained on the differences in the effects on bone marrow progenitor cells, neutrophil response, detection of antivirus antibodies, and human leucocyte antigen typing, but no significant differences were observed in most of the study results, for example the plasma concentrations of unchanged drug and its metabolites, blood cytokine concentration, lymphocyte stimulation test, effects on neutrophils, and the detection of autoantibodies and several antibodies against bone marrow hemopoietic progenitors, anti-granulocyte, anti-neutrophil, and antidrug antibodies. Consequently, this information indicates that the conclusive mechanism has not yet been identified. Several mechanisms of IDT are hypothesized; however, because of their idiosyncratic nature, it is exceedingly difficult to perform mechanistic studies of IDT to test these hypotheses (Johnston and Uetrecht, 2015). In particular, the delay in onset is characteristic of an immune-mediated reaction, presumably because it takes time for the few T cells with the required specificity to proliferate and for those with the highest affinity to become dominant (Uetrecht and Naisbitt, 2013). However, immune-mediated reactions were not clearly detected using patient samples.
In this study, we focused on the MPO metabolism of vesnarinone and attempted to identify the reactive metabolites that induce cytotoxicity in HL-60 cells. Furthermore, it is known that oxidative stress is involved in various diseases, and GSH, an endogenous antioxidant, is involved in that defense (Mastronikolis et al., 2022). There was a clinical report that blood GSH levels were decreased in patients with agranulocytosis who were treated with phenylbutazone (Kuzell et al., 1955), suggesting that oxidative stress in patients may contribute to the development of agranulocytosis. Therefore, we also examined the protective effect of GSH on vesnarinone cytotoxicity.
Vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxy-benzoyl)-1-piperazinyl]-2(1H)-quinolinone) and its metabolites, M1: Metabolite 1 (4-(3,4-dimethoxybenzoyl)piperazine) and M2: Metabolite 2 (1-chloro-4-(3,4-dimethoxybenzoyl)piperazine), were synthesized by Otsuka Pharmaceutical Co., Ltd. MPO and RPMI-1640 medium containing L-glutamine and sodium bicarbonate were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) H2O2, 2’,7’-dichlorofluorescin diacetate (DCFDA), and L-buthionine-(S, R)-sulphoximine (BSO) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biowest (St. Louis, MO, USA). Bovine catalase, GSH, and oxidized GSH (GSSG) were purchased from Sigma (Tokyo, Japan).
All other chemicals and solvents were of analytical grade or the highest grade commercially available. All test substances were dissolved in DMSO, with the exception of BSO and GSH, which were dissolved in RPMI-1640 medium containing 2% FBS (hereinafter, referred to as 2% FBS medium).
HL-60 cell cultureHL-60 cells (DS Pharma Biomedical Co., Ltd., Osaka, Japan) were grown in RPMI-1640 medium containing 20% heat-inactivated FBS at 37°C in an atmosphere of 5% CO2. For the cytotoxicity experiments, HL-60 cells were cultured in 2% FBS medium at 37°C and 5% CO2.
Treatment of HL-60 cellsHL-60 cells (2 × 104 cells/well) were seeded into clear-bottomed 96-well plates and treated with vesnarinone (3.3–100 µM) at 37°C in an atmosphere of 5% CO2 for 24 or 48 hr. To deplete intracellular GSH, HL-60 cells were treated with 10, 50, or 100 μM BSO for 24 or 48 hr. Before HL-60 cells were exposed to the vesnarinone–MPO/H2O2/Cl− reaction mixture, vesnarinone was reacted with MPO/H2O2/Cl− for 0.5 hr in advance, and then an aliquot (10%) of the reaction mixture was exposed to the HL-60 cells for 24 hr in the absence or presence of GSH (1 mM). The conditions used for reaction with MPO/H2O2/Cl− were as follows: MPO: 4 units, H2O2: 0.44 mM, 0.1 M phosphate buffer (pH 7.4) containing 5 mM MgCl2, and vesnarinone: 100 μM. Furthermore, to attenuate the effect of H2O2, HL-60 cells were treated with catalase (0.1 mg/mL) just prior to exposure to the MPO reaction mixture. The test solutions of synthesized M1 (100 µM) and M2 (3.3, 10, and 33 µM) were incubated with HL-60 cells in a 96-well plate for 24 hr in the absence or presence of GSH (1 mM).
Cytotoxicity assay (CCK-8 assay)Cytotoxicity was assayed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) and water-soluble 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8). WST-8 produces a water-soluble formazan dye upon reduction in the presence of an electron carrier coupled with mitochondrial dehydrogenases. HL-60 cells (2 × 104 cells/well) were seeded into 96-well plates. After incubation, 10 µL of CCK-8 reagent was added to the culture medium (100 µL), and the absorbance of WST-8 formazan was measured at 450 nm at room temperature. The percentage of viable cells was calculated relative to the absorbance in the control wells.
GSH assayGlutathione levels were measured using a luminometric GSH kit (GSH-Glo™ Assay, Promega; Madison, Wisconsin, USA). HL-60 cells (2 × 104 cells/well) were seeded into 96-well plates for 24 or 48 hr in an incubator (37°C in 5% CO2). After incubation, Luciferin Detection Reagent was added 100 μL to all wells and equilibrated for 15 min. A luminometer (Varioskan Flash Multimode Reader, Thermo, Waltham, MA, USA) was used to measure GSH levels in cells. Luminescence emitted from the wells is directly proportional to glutathione. The results were calculated as a percentage of the luminescence relative to the control.
Oxidative stress measurement (ROS assay)ROS production was estimated by measuring DCF fluorescence. The cells (2 × 104 cells/well) were seeded in 96-well plates. After incubation for 48 hr, the medium was removed and the cells were rinsed with PBS. For each treatment, cells from 12 wells were collected and separated into 3 wells, then incubated with 5 μM DCFDA for 0.5 hr in a CO2 incubator. The DCF fluorescence resulting from the interaction of the dye with ROS was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
Liquid chromatography–mass spectrometry (LC–MS) analysis of the vesnarinone–MPO/H2O2/Cl− reaction mixtureBecause it was suggested that the cytotoxicity caused by vesnarinone was a result of metabolism by MPO, this reaction mixture was analyzed by LC–MS. Furthermore, the reaction under conditions of 1 mM GSH was investigated. Each reaction mixture was deproteinized with twice the volume of acetonitrile and its centrifugal supernatant was used as a measurement sample. Each aliquot of the supernatant was injected into a Quattro Micro API LC–MS system (Waters, Milford, MA, USA) equipped with an electrospray ionization interface and an ultraviolet detector (wavelength at 254 nm). Metabolites were separated on an AQUASIL C18 column (150 × 2.1 mm, 3 µm; Thermo Fisher Scientific, Waltham, MA, USA). The gradient mobile phase consisted of purified water containing 0.1% formic acid and 5% acetonitrile and 0.1% formic acid in acetonitrile (100:0 to 0:100, v/v). The mobile phase was eluted at 0.2 mL/min using a sample organizer pump (Waters). Vesnarinone and its metabolites, GSH, and GSSG were identified by comparing their retention times and spectra with those of synthesized or purchased compounds (data not shown). Analytical data were processed using MassLynx software, version 4.1 (Waters).
Statistical analysesData are expressed as the mean ± standard deviation (S.D.) of three independent determinations. The statistical significance of the cytotoxicity data was determined using the two-tailed Student’s t-test (Microsoft Office Excel 2013). A value of p < 0.05 was considered statistically significant.
First, the effect of vesnarinone on the viability of HL-60 cells was determined. The viability of HL-60 cells did not change up to 100 μM vesnarinone in 24 hr treatment. After 48 hr of the treatment, a slight tendency for the viability to decrease with 100 μM vesnarinone was observed, but no clear concentration-dependent cytotoxicity was showed. (Fig. 1A). However, when the GSH content in HL-60 cells was examined, it decreased significantly after 48 hr of vesnarinone treatment (Fig. 1B). These results suggest that exposure to vesnarinone decreased GSH content, which causes oxidative stress within the cells.
Cell viability and GSH content of HL-60 cells after exposure to vesnarinone. HL-60 cells (2 × 104 cells/well) were exposed to vesnarinone (Ves) in a 96-well plate for 24 or 48 hr. Cell viability (A) and GSH content (B) were assessed by the CCK-8 and GSH assays, respectively. Each point represents the mean ± S.D. of triplicate determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with each control treatment (0 µM).
To evaluate the effect of vesnarinone under conditions where the GSH content in HL-60 cells was reduced in advance, BSO treatment conditions were investigated. When treated 10, 50, and 100 μM BSO for 24 or 48 hr, the intracellular GSH content rapidly decreased to 19.1%–31.2% at 24 hr and 10.8%–20.5% at 48 hr (Fig. 2A). Cytotoxicity was also confirmed at the same time, and no clear cytotoxicity was observed after 24 hr treatment, but significant cytotoxicity was observed with 50 and 100 μM BSO after 48 hr (Fig. 2B). Therefore, it was found that the optimal concentration of BSO was 10 μM for a 48-hr treatment.
Investigation of BSO treatment conditions in HL-60 cells. BSO was exposed to HL-60 cells (2 × 104 cells/well) in a 96-well plate for 24 or 48 hr. GSH content (A) and cell viability (B) were assessed by the GSH and CCK-8 assays, respectively. Each point represents the mean ± S.D. of triplicate determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with each control treatment (0 µM).
Next, we examined whether vesnarinone affects the viability of HL-60 cells under GSH depletion conditions. When HL-60 cells were exposed to vesnarinone after pretreatment with BSO (10 μM), cell viability after exposure for 48 hr significantly decreased in a concentration-dependent manner (Fig. 3A). When ROS production associated with vesnarinone treatment was investigated, ROS production due to BSO treatment was also significantly detected in a concentration-dependent manner (Fig. 3B). Therefore, vesnarinone exposure caused oxidative stress and exerted cytotoxic effects in HL-60 cells.
Cell viability and ROS production in HL-60 cells treated with vesnarinone under GSH-depleting conditions. Vesnarinone (Ves) was exposed to HL-60 cells (2 × 104 cells/well) in a 96-well plate for 48 hr in the absence or presence of BSO (10 μM). Cell viability (A) and ROS production (B) were assessed by the CCK-8 and ROS assays, respectively. The symbols of GSH (-) and GSH (+) mean no GSH treatment and GSH treatment, respectively. Each point represents the mean ± S.D. of triplicate determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with each control treatment (0 µM). ##p < 0.01 and ###p < 0.001 compared between no BSO treatment and BSO treatment.
After preparing a reaction mixture in which vesnarinone (100 μM) was metabolized with MPO for 0.5 hr, HL-60 cells were exposed to an aliquot of the reaction mixture to examine whether similar cytotoxicity was observed. When comparing between the absence and the presence of GSH (1 mM) in the medium, cytotoxicity was found in HL-60 cells in the absence of GSH and the protection effect of GSH against cytotoxicity was also observed (Fig. 4). This result was similar to that obtained when vesnarinone was exposed to GSH-depleted HL-60 cells.
Viability of HL-60 cells treated with a vesnarinone–MPO/H2O2/Cl− reaction mixture. After vesnarinone was metabolized by MPO/H2O2/Cl− for 0.5 hr, an aliquot of the reaction mixture was exposed to HL-60 cells (2 × 104 cells/well) in a 96-well plate for 24 hr in the absence or presence of GSH (1 mM). Cell viability was assessed by the CCK-8 assay. The symbols of GSH (-) and GSH (+) mean no GSH treatment and GSH treatment, respectively. Exp. MPO (+) refers to the exposure of the vesnarinone–MPO/H2O2/Cl− reaction mixture and Exp. MPO (-) refers to the non-exposure of that. Each point represents the mean ± S.D. of triplicate determinations. ***p < 0.001 compared with each Exp. MPO (-) treatment.
In the search for metabolites in the vesnarinone–MPO/H2O2/Cl− reaction mixture, M1 (retention time, RT: 4.4 min) and M2 (RT: 9.6 min) were detected in addition to vesnarinone (RT: 9.0 min) (Fig. 5A). In the presence of GSH (1 mM), the M2 peak disappeared and the M1 peak increased; moreover, although peak separation was not possible, the added GSH (RT: 2.2 min) and produced GSSG (RT: 2.2 min) could also be detected (Fig. 5B). Structural analysis was performed using the obtained MS spectra (data not shown). GSH ([M+H]+: m/z 308) and GSSG ([M+H]+: m/z 613) were detected. By comparison with the spectra of synthetic compounds, M1 ([M+H]+: m/z 251) and M2 ([M+H]+: m/z 285) were identified as 1-(3,4-dimethoxybenzoyl)piperazine and 1-chloro-4-(3,4-dimethoxybenzoyl)piperazine, respectively.
LC–MS chromatograms of the vesnarinone–MPO/H2O2/Cl− reaction mixture. After HL-60 cells were exposed to the vesnarinone–MPO/H2O2/Cl− reaction mixture in the absence (A) or presence (B) of GSH (1 mM), the culture medium was immediately collected, centrifuged, and subject to protein removal. The supernatant, dissolved in two volumes of acetonitrile, was subjected to LC–MS analysis. The UV wavelength was set at 254 nm.
Finally, HL-60 cells were exposed to the synthesized metabolites, M1 and M2, to examine their cytotoxicity. At 100 µM of M1, no cytotoxicity was observed (Fig. 6A), but concentration-dependent and significant cytotoxicity was observed after exposure to M2 (3.3, 10, and 33 μM) (Fig. 6B). Moreover, the cytotoxic effects of M2 were perfectly counteracted by the addition of GSH (1 mM). These results suggest that M2, not M1, is the main cause of cytotoxicity.
Cell viability of synthetic M1 and M2 in HL-60 cells. The synthetic M1: Metabolite 1 (A) and M2: Metabolite 2 (B) were incubated with HL-60 cells (2 × 104 cells/well) in a 96-well plate for 24 hr and in the absence or presence of GSH (1 mM). Cell viability was assessed using the CCK-8 assay. Each point represents the mean ± S.D. of triplicate determinations. *p < 0.05 and ***p < 0.001 compared with each control treatment (0 µM).
In repeated-dose toxicity studies of vesnarinone in rats and dogs, no significant hematological toxicities were noted (data on file, 1996). However, when clinical trials were initiated in the United States, 4 of the first 28 patients developed agranulocytosis (Uetrecht, 1996). Thus, these cases of agranulocytosis caused by vesnarinone were thought to be due to the human-specific toxicity of the drug.
Therefore, we used HL-60 cells, which are commonly used as a model of neutrophil precursor cells, to examine the cytotoxic effects of vesnarinone. We found that almost no cytotoxicity was observed around the mean clinical serum concentrations (24 μM, repeated dose of 60 mg/day for 4 weeks) (Fig. 1A). Interestingly, although little cytotoxicity was observed up to 100 μM (Supplementary Fig. 1), a significant decrease in intracellular GSH content was observed after vesnarinone treatment for 48 hr (Fig. 1B). Furthermore, by adjusting conditions to have reduced intracellular GSH (Fig. 2A and 2B), we were able to detect the cytotoxicity of vesnarinone as well as ROS production (Fig. 3A and 3B). In the case of patients with agranulocytosis treated with phenylbutazone, this phenomenon was thought to be consistent with a decrease in blood GSH levels. Furthermore, when we reevaluated the cytotoxicity of vesnarinone in a state where reduced GSH was predominant in HL-60 cells, to mimic a patient with reduced GSH, concentration-dependent cytotoxicity was observed. Myeloperoxidase is a heme-containing enzyme that is present in mature neutrophils and many myeloid progenitor cells and is well known for its role in inflammation and host defense against several pathogens (Aratani, 2018). Therefore, it has been suggested that in humans, there may be an increased risk of cytotoxicity, not only with vesnarinone administration but also in patients with potentially decreased GSH content in neutrophils and myeloid progenitor cells.
As shown in Supplementary Fig. 2 (B), HL-60 cells exposed to vesnarinone after GSH depletion were subjected to LC–MS analysis, and M1 was observed in addition to vesnarinone. It was suggested that the oxidation reaction may be caused by MPO present in HL-60 cells. Indeed, when catalase, which can decompose hydrogen peroxide, was present in the test system, the cytotoxicity mentioned above was attenuated (Supplementary Fig. 3), suggesting the involvement of MPO. To verify this, we exposed HL-60 cells to a reaction mixture of vesnarinone metabolized by MPO/H2O2/Cl− and observed cytotoxicity in GSH-free conditions (Fig. 4). Moreover, when this reaction mixture was analyzed by LC–MS, M1 was similarly detected, and M2 was also detected for the first time (Fig. 5A). M2 is a new metabolite that has not previously been reported. Interestingly, it was revealed that M2 was converted to M1 in the presence of GSH (Fig. 5B). Furthermore, studies using synthetic compounds have confirmed that M2 was converted to M1 in the presence of GSH, accompanied by GSSG production by MS spectrum analysis (data not shown). In the case of drugs other than vesnarinone, agranulocytosis induced by ticlopidine and clozapine was thought to be a possible mechanism of detoxification by GSH (Aizawa et al., 1997; Pereira and Dean, 2006). Again, a decrease in the GSH level in the whole blood was reported following treatment with phenylbutazone in a patient with agranulocytosis and anemia (Kuzell et al., 1955). A greater sensitivity of cells in some patients, related to age, diet, enzyme induction, or genetic factors, may allow toxic metabolites to escape inactivation (Uetrecht, 1996).
By comparing the chromatograms of the metabolic mixture of vesnarinone in the absence or presence of GSH, M2 was identified as 1-chloro-4-(3,4-dimethoxybenzoyl)piperazine, an N-chloramine of M1, and determined to be the causal metabolite of cytotoxicity (Fig. 5A and 5B). In addition, the CCK-8 assay revealed the cytotoxicity of M2 and the protective effect of GSH (Fig. 6A and 6B). N-chloramines easily react with thiol groups and generate GSSG by reacting with GSH (Thomas et al., 1985). In addition, they can mediate protein damage and induce the oxidation of other biological molecules, including lipids (Hazell et al., 1999) and DNA (Hawkins et al., 2002), without accumulating at detectable concentrations (Thomas et al., 1983). Therefore, the cytotoxicity observed in Fig. 6B was thought to be due to direct damage to the cell membrane from externally added N-chloramine M2. This is because, under conditions where intracellular GSH is not depleted, it is assumed that M2 that has permeated the membrane is converted to M1. Indeed, GSH was easily oxidized to GSSG when added to the metabolic mixture of vesnarinone (Fig. 5B). Thus, we hypothesized that the possible mechanism for vesnarinone-induced agranulocytosis is as described in Fig. 7. In brief, vesnarinone reacts with MPO/H2O2/Cl− (HClO), generating M1 and M2. If GSH is sufficiently abundant in neutrophils or bone marrow precursor cells, M2 readily reacts with GSH and is converted into M1 and GSSG. If not, M2 reacts with biological molecules (e.g., thiol groups), resulting in the development of cytotoxic effects.
Proposed scheme for vesnarinone-induced cytotoxicity in HL-60 cells.
Bertolet et al. (1994, 2004) reported seasonal variation in vesnarinone-induced agranulocytosis and suggested a possible association with influenza vaccine or infection because more patients develop this type of toxicity in the fall and winter seasons. Therefore, factors such as vaccination or infection may activate neutrophils, increase the number of reactive metabolites (e.g., iminium ion, quinone imine) (Uetrecht et al., 1994), decrease the amount of GSH, and induce agranulocytosis. GSH adducts of the iminium ion and quinone imine derived from vesnarinone were not detected in the chromatograms. It is unclear exactly why these adducts were not detected, but N-chloramine may have been detected because of its relatively long half-life.
Several recent studies report the possibility that GSH is involved in the detoxification mechanism of agranulocytosis. N-methyl-4-aminoantipyrine (MAA), the principal metabolite of metamizole, increased hemin cytotoxicity by a reaction involving the formation of an electrophilic metabolite. These findings indicate that the cytotoxicity of MAA/hemin could be prevented by the iron chelator EDTA and the electron donor GSH (Rudin et al., 2019). In the case of clozapine, its reactive metabolite decreased the levels of GSH, whereas the addition of GSH attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that GSH metabolism plays a role in hematopoietic toxicity and oxidative stress (Torii-Goto et al., 2022).
In conclusion, we have demonstrated the cytotoxicity and ROS production associated with vesnarinone originate through the MPO/H2O2/Cl− metabolic reaction and newly identified the reactive metabolite, M2, which is the N-chloramine form of M1 that induces cytotoxicity. The addition of GSH was protective against the cytotoxicity of M2, converting it to nontoxic M1. Therefore, it was suggested that GSH may play an important role and the degree of oxidative stress among patients (especially in neutrophils and myeloid progenitor cells) may be a factor driving the onset of agranulocytosis.
We would like to thank Dr. Toyoki Mori and Dr. Hiroyuki Sasabe for their scientific advice and MARUZEN-YUSHODO Co., Ltd. (https://kw.maruzen.co.jp/kousei-honyaku/) for English language editing.
Conflict of interestThe authors declare that there is no conflict of interest.
